Method Article

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

DOI:

10.3791/56069

⸱

August 8th, 2017

 , 
1Department of Developmental Biology, Stanford University School of Medicine, 2Department of Chemical and Systems Biology, Stanford University School of Medicine

In This Article

Summary

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This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

Abstract

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The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that >1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

Introduction

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Biological systems frequently experience transient fluctuations in protein abundance. Whether these have a lasting impact in shaping the phenotype of an organism or of future generations remains unclear. The best-known instances of this biology involve a rare class of proteins, prions, which drive the emergence of heritable traits without genome modification. Instead, these proteinaceous and infectious particles transmit phenotypes via self-perpetuating changes to protein conformation1,2. This type of inheritance was discovered as the cause of the unusual inheritance patterns of a devastating neurodegenerative disease. However, studies in organisms ranging from fungi to mammals3,4,5,6,7,8,9,10 have since revealed that prion-like elements can confer adaptive value. Nonetheless, prions have been viewed as a fascinating but rare biological oddity.

This prevailing wisdom is in part held because the characterization of protein-based inheritance has long been restricted by a small set of examples. Recent systematic screening efforts have widened this picture significantly by identifying several new bona fide prions11 and almost two dozen protein domains12 with the capacity to fuel prion-like conformational conversion. However, because these approaches have generally focused on strong amino acid sequence biases, the prions that have been discovered share the biochemical properties of the founding yeast prions [PSI+]13,14, [URE3]15, and [RNQ+]11,16. These include: 1) modular domains that are rich in long polymeric stretches of asparagine (N) and glutamine (Q), 2) assembly into an amyloid [PRION+] conformation17,18,19, and 3) complete reliance on disaggregase Hsp104 function for faithful propagation from mother to daughter13,20,21. Indeed, many bona fide prions, including [GAR+], [Het-s], and even the original prion (PrPSc), would be missed under such stringent criteria. Perhaps more importantly, they would be unable to capture any novel mechanisms of protein-based inheritance22. Thus, the true biological breadth of such phenomena may be far more common in nature than previously assumed.

To investigate this question, a high-throughput, proteome-wide strategy was employed. A hallmark of all prions, including PrPSc, [GAR+], and [Het-s], is that the transient overexpression of the causal proteins strongly increases the rate of prion acquisition15,23,24,25,26. We took advantage of this feature to systematically ask, across the entire yeast ORFeome, if stable protein-based, epigenetic states could be initiated by transiently inducing the overexpression of individual proteins. It is well known that protein overexpression can alter phenotypes27. However, prion proteins are unusual because their temporary overproduction produces a change in phenotype that is heritable for many hundreds of generations after the initial overexpression. We previously took advantage of this feature, as well as the unusual inheritance patterns of protein-based genetic elements, to identify dozens of proteins that are capable of heritably re-wiring phenotypic landscapes without altering the genome28. Although some identified proteins were previously known as prions, most were not, underscoring the power of this approach to uncover new forms of protein-based inheritance.

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Protocol

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1. Initial Overexpression

  1. Transform the yeast cells (in this case, BY4741 MAT a haploids) previously grown in YPD liquid (10 g of yeast extract, 20 g of peptone, and 20 g of glucose per 1 L) with the desired candidate constructs from the FLEXGene ORFeome library (yeast ORFs under the control of a galactose-inducible promoter in the URA3-marked centromericplasmid backbone, pBY01129).
  2. Use autoclaved toothpicks to pick four separate colonies from these transformations to serve as biological replicates. Grow them for 48-72 h in 150 µL of SRaffinose-URA (0.74 g of CSM-URA, 6.7 g of yeast nitrogen base without amino acids, and 20 g of raffinose per 1 L) in 96-well plates. Inoculate colonies from the first ORF in well A1 of all plates, colonies from the second ORF in A2 of all plates, etc.
    NOTE: All growth steps are conducted at 30 °C and at atmospheric levels of CO2 (407.05 ppm), unless stated otherwise.
  3. Confirm that the cultures are saturated (cells visible by eye at the bottom of each well) and use a liquid handling robot to inoculate a 1:4 array of 1 µL from each 96-well plate well into 4 separate wells of a 384-well plate filled with 45 µL of SGal-URA (0.74 g of CSM-URA, 6.7 g of yeast nitrogen base without amino acids, and 20 g of galactose per 1 L) per well.
    NOTE: This creates a composite plate in which 4 biological replicates of each ORF are arrayed in a square pattern.
  4. At the same time, prepare separate 384-well plates containing 45 µL of SGal-URA with the stressors of interest (e.g., manganese chloride at 20 mM) per well. Inoculate this plate in the same manner as in step 1.3.
    NOTE: To achieve the greatest dynamic range for phenotypic detection, a 2-fold dilution series around the LD50 concentration of a given stressor is recommended. This can be done beforehand to determine the optimum concentration at which to observe both enhanced and reduced growth.
  5. As a final parallel control, inoculate a third 384-well plate in the same manner, with the same stressor included in medium that will not induce plasmid expression. Ensure that the plate contains 45 µL of SD-URA (0.74 g of CSM-URA, 6.7 g of yeast nitrogen base without amino acids, and 20 g of glucose per 1 L) per well and is inoculated as in steps 1.3 and 1.4.
  6. Immediately place plates with cells on a microplate stacker and set the protocol for a 72-h continuous loop, measuring the OD600 with a microplate reader equipped with stackers at room temperature and atmospheric CO2 (407.05 ppm).
    Note: The frequency of the growth measurement will depend on the number of plates (ultimately determined by the number of genes and conditions the investigator wants to probe) used in the run. The more plates used, the longer it will take the plate reader to complete a loop and thus the longer time between measurements (measurement time per plate is ~45 s, depending on the instrumentation employed).
  7. After the growth measurements, use the same liquid handling robot to transfer 1 µL per well of the SGal-URA-induced cultures (i.e., those that experienced protein overexpression) to new 384-well plates containing 45 µL of SD-URA per well (medium that does not permit protein expression of the plasmid).
    1. In parallel, perform analogous inoculations of a second set of 384-well plates containing 45 µL of SD-URA per well from the cultures that were grown in SD-URA in the presence of the stressors (i.e., those from step 1.5).
    2. Grow the plates for 48 h at 30 °C to saturation in a humidified chamber (i.e., a sealable plastic bin with a damp paper towel inside).
  8. Take the plates from the previous step and reinoculate 1 µL per well in 384-well plates containing 45 µL of SD-URA. Then, perform a separate reinoculation of 1 µL per well from the same source plate into a separate 384-well plate containing 45 µL of SD-URA with the stressor.
    NOTE: This will determine if the sensitivity/resistance phenotypes to a stressor persist after the protein overexpression has ceased.
  9. Immediately place plates with cells on a microplate stacker and set the protocol for a 48-h continuous loop measuring the OD600 with a microplate reader at room temperature and atmospheric CO2 (407.05 ppm).
  10. Export the time versus OD600 measurements as an XY table using the plate reader software. Group columns of the OD600 for each biological replicate together and calculate the mean. Create an XY plot of time versus OD600 to generate growth curves.
    1. Compare the growth rates of cultures that had been subjected to a past overexpression (i.e., those originally induced in galactose-containing medium before reinoculation in SD-URA) versus cultures that had not (i.e., those propagated in glucose), as described previously30.
  11. For cultures that show significant growth differences in response to a given stressor, dependent upon ancestral protein overexpression, take 1 µL from each biological replicate, dilute it in 10 mL of water, and then plate 50 µL on plates containing 5-FOA (0.74 g of CSM-URA, 1 g of 5-FOA, 50 mg of uracil, 6.7 g of yeast nitrogen base without amino acids, 20 g of glucose, and 20 g of agar per liter). Grow at 30 °C for 3 days.
    1. If this results in too many or too few colonies per plate, adjust the dilution factor accordingly.
      Note: 5-FOA is converted into a toxic intermediate by the uracil biosynthesis pathway. This will cause a loss of the URA3-marked expression plasmid transformed in the first step.
  12. Pick 8-32 single colonies with autoclaved toothpicks and pin them to 96-well plates containing 150 µL of SD-CSM. Grow to saturation for 48-72 h in a humidified chamber at 30 °C. Use these cultures to inoculate two new sets of 96-well plates containing 150 µL of SD-CSM, both with and without the stressors from step 1.11.
  13. Place the plates on a microplate stacker and set the protocol to measure the OD600 on a 48-h continuous loop.
  14. Analyze the data as in step 1.10 and confirm that the significant growth differences seen in step 1.10 are maintained after plasmid loss.
    NOTE: These cells harbor stable phenotypic states that might be indicative of protein-based inheritance.
  15. Test the cells that maintained the induced phenotypes for classic hallmarks of protein-based inheritance (see below).

2. Tests for Prion-like Inheritance

  1. Chaperone-mediated "curing"
    1. Test Hsp104 chaperone dependence.
      1. Pick a colony of a yeast strain harboring a stable phenotypic state, streak to single colonies on a YPD plate (10 g of yeast extract, 20 g of peptone, 20 g of glucose, and 20 g of agar per 1 L) containing 3 mM GdnHCl and grow at 30 °C for 3 days. In parallel, streak out a colony from a naïve strain to single colonies on YPD+ 3 mM GdnHCl as a control in the same manner.
      2. Repeat 2 more times on fresh YPD plates containing 3 mM GdnHCl.
      3. Because GdnHCl exposure can increase the frequency at which petite cells arise, pick multiple colonies that have undergone 3 passages on GdnHCl and check for functional mitochondrial respiration by examining their ability to grow into visible colonies on YP-Glycerol plates (10 g of yeast extract, 20 g of peptone, 20 mL of glycerol, and 20 g of agar per 1 L) after 7 days.
      4. Pick multiple colonies from the YP-Glycerol plates and test for the maintenance of stable phenotypic states using growth in the presence of the stressor compared to non-"cured" and naïve (i.e., isogenic cells that do not harbor the phenotypic state, such as BY4741 MATa haploids) controls in a similar manner as described in step 1.13.
    2. Test Hsp70 chaperone dependence.
      1. "Cure" via a plasmid.
        1. Transform both naïve cells and cells harboring stable phenotypic states with URA3-marked plasmids expressing a dominant negative allele of Hsp70 (K69M)25,31 from a strong constitutive promoter (GPD).
        2. Pick single transformants for each, streak to single colonies on an SD-URA plate (0.74 g of CSM-URA, 6.7 g of yeast nitrogen base without amino acids, 20 g of glucose, and 20 g of agar per 1 L), and grow for 3 days at 30 °C and atmospheric CO2.
        3. Repeat this passaging 2 more times on SD-URA plates.
        4. Pick multiple colonies and streak to single colonies on 5-FOA plates to eliminate the plasmid.
        5. Pick multiple single colonies for each isolate and test for retention (or loss) of the stable phenotypic states in a similar manner as described in step 1.13.
      2. "Cure" via genetic crossing.
        1. Cross BY4741 MATa haploid strains harboring stable phenotypic states and naïve strains of the opposite mating type (in this case, BY4742 MATα) harboring genetic deletions in two of the four yeast Hsp70 paralogs (ssa1Δ ssa2Δ).
          NOTE: This strain is deficient in Hsp70 chaperone function.
        2. Select for the growth of diploids by streaking to single colonies on SD-LYS-MET (0.74 g of CSM-LYS-MET, 6.7 g of yeast nitrogen base without amino acids, 20 g of glucose, and 20 g of agar per 1 L) double-dropout medium.
        3. Pick single diploid colonies and grow for 24 h at 30 °C in 8 mL of pre-sporulation medium (0.8% yeast extract, 0.3% peptone, 10% dextrose, and 100 mg/L adenine sulfate).
        4. Spin down the cultures for 3 min at 3,000 xg, aspirate the pre-sporulation (pre-SPO) medium, and wash the pellets once with sterile water.
        5. Re-suspend the pellets in 2 mL of sporulation medium (1% potassium acetate, 0.1% yeast extract, 0.05% glucose, and 0.01% amino acid add-back mix (2 g of histidine, 10 g of leucine, 2 g of lysine, and 2 g of uracil)). Grow at 25°C for 5 days.
        6. Move the cultures to 30 °C and incubate for an additional 48 h.
        7. Assess the efficiency of sporulation by looking for the presence of tetrads using a light microscope32.
        8. Take 40 µL of culture, spin down for 1 min at 3,000 xg in a 1.5-mL tube, and re-suspend in an equal volume of zymolyase enzyme mix (1 M sorbitol, 0.1 M EDTA, and 10 mg/mL zymolyase 100T)
        9. Incubate at 25 °C for 5 min.
        10. Apply 10 µL of digested culture to a very dry, very level YPD agar plate and allow the culture to slowly spread down the plate in a straight line.
        11. Allow the plates to dry for at least 30 min in a laminar flow hood.
        12. Using a dissection microscope, separate the spores into tetrads and array them on the plate.
        13. Allow the individual haploid spores to grow into colonies and test for their ability to grow on SD-HIS-LEU (0.67 g of CSM-HIS-LEU, 6.7 g of yeast nitrogen base without amino acids, 20 g of glucose, and 20 g of agar per 1L) plates, indicating that they harbor both genetic deletions (ssa1Δ ssa2Δ).
        14. Test each for the maintenance of the phenotypic state in the Hsp70-deficient background in a similar manner as described in step 1.13.
          NOTE: Crossing these spores back to a wildtype strain with restored Hsp70 function should not restore the prion phenotype.
  2. Testing for non-Mendelian inheritance.
    1. Cross BY4741 MATa haploid strains harboring stable phenotypic states and isogenic naïve controls to an isogenic naïve strain of the opposite mating type (in this case, BY4742 MATα).
    2. Select for the growth of diploids by streaking to single colonies on SD-LYS-MET double-dropout medium.
    3. Pick single diploid colonies and grow for 24 h at 30 °C in 8 mL of pre-sporulation medium.
    4. Repeat steps 2.2.2.3-2.2.2.12, as described above.
    5. Allow the individual haploid spores to grow into colonies and test each for maintenance of the phenotypic state through meiosis using the growth assay, as in step 1.13.
      Note: Another defining feature of prions is that they can be heritably eliminated through the removal of the original casual protein. Thus, analogously crossing a prion strain with a strain harboring a genetic deletion of the original induced protein will abrogate prion phenotypes in the spores that inherit the deletion. Also note that is possible for the prion to be maintained in a "cryptic" state in such a mutant if the deleted gene is required to manifest the prion phenotype but not to propagate the prion itself. To distinguish between these two possibilities, cross the spores back to a naïve wildtype strain and test whether the prion phenotype re-emerges in a diploid genetic background, where the function of the gene has been restored.
  3. Cytoduction.
    1. Generate the initial BY4742 recipient strain.
      1. Through transformation, introduce a defective KAR allele (kar1-15) that prevents nuclear fusion during mating23.
      2. Make this strain "petite" (i.e., incompetent for mitochondrial respiration) by inoculating a single colony in YPD broth with 0.25% ethidium bromide.
      3. Grow the culture at 30 °C until late exponential/stationary phase (OD600 ~1).
      4. Dilute 1:1,000 in fresh YPD with 0.25% ethidium bromide and repeat twice.
      5. Once the culture reaches late exponential/early stationary phase (OD600 0.8-1.2), plate to single colonies by diluting 1:10,000 in sterile water and plating 50 µL on a YPD plate. After growing for 3 days at 30 °C, pick multiple colonies and test each for respiration incompetence by examining their ability to grow into visible colonies on YP-Glycerol plates after 7 days.
    2. Perform initial cytoduction into BY4742.
      1. Mix cells of the donor BY4741 strain harboring a stable phenotypic state with cells of the naïve BY4742 kar1-15 recipient strain on the surface of a YPD agar plate.
      2. Grow for 24 h at 30 °C and atmospheric CO2 (407.05 ppm) and transfer to methionine dropout medium containing glycerol (SGly-MET: 0.74 g of CSM-MET, 6.7 g of yeast nitrogen base without amino acids, 20 mL of glycerol, and 20 g of agar per 1 L).
        NOTE: This selects for both BY4742 nuclear markers, along with allowing the restoration of functional mitochondria via cytoplasmic exchange.
      3. After 3-5 days, pick multiple single colonies and perform another round of selection on SD-MET (0.74 g of CSM-MET, 6.7 g of yeast nitrogen base without amino acids, 20 g of glucose, and 20 g of agar per 1 L).
      4. In parallel, confirm that the colonies are not diploids by passaging on SD-LYS-MET medium.
    3. Perform reverse cytoductions.
      1. Mix the new donor strains (this time, the successful BY4742 kar1-15 cytoductants) with petite naïve BY4741 cells (generated as above) on YPD agar.
      2. Repeat cytoductions, as described previously (steps 2.3.2.1-2.3.2.4), except selecting for BY4741 recipient nuclear markers on glycerol medium lacking lysine (SGly-LYS: 0.74 g of CSM-LYS, 6.7 g of yeast nitrogen base without amino acids, 20 mL of glycerol, and 20 g of agar per 1 L).
      3. Pick multiple cytoductants and test for the maintenance of stable phenotypic states using the growth assay, as before in step 1.13.
  4. Protein transformation.
    1. Prepare the lysate.
      1. Grow 50 mL of cells harboring stable phenotypic states in YPD for 18 h at 30 °C.
      2. Pellet the cultures at 3,000 x g for 4 min and then wash twice: once in autoclaved H2O and then in 1 M sorbitol.
      3. Re-suspend the cells in 200 mL of SCE buffer (1 M sorbitol, 10 mM EDTA, 10 mM DTT, 100 mM sodium citrate, and 1 mini-EDTA-free protease inhibitor tablet per 50 mL, pH 5.8) with 50 U/mL of yeast lytic enzyme 100T.
      4. Incubate the mix at 35 °C for 30 min.
      5. Sonicate the cells at 20 kHz and 20% intensity for 10 s on ice with a sonic dismembrator.
      6. Remove the cell debris by centrifugation at 10,000 x g for 15 min at 4 °C.
      7. Move the supernatants to a clean tube and add RNase I and biotinylated DNase at a 3-fold excess (as determined by units of activity) and incubate at 37 °C for 1 h.
      8. Remove the DNase I by adding excess streptavidin-agarose beads, incubating for 5 min, and pelleting by centrifugation at 10,000 x g for 1.5 min.
      9. Transfer the supernatant to a fresh tube.
    2. Prepare the recipient spheroplast.
      1. Grow 5 mL of naïve recipient cells in YPD to mid-exponential phase (OD600 ranging from 0.5-1).
      2. Harvest by centrifugation (3,000 x g for 4 min) and wash 4 times: twice in H2O and twice in 1 M sorbitol.
      3. Re-suspend the cells in 1 M sorbitol containing 200 U/mL yeast lytic enzyme and incubate for 15 min at 35 °C to digest the cell walls.
      4. Harvest the resulting spheroplasts with gentle centrifugation at 600 x g for 5 min and wash with 1 mL of 1 M sorbitol and then again with 1 mL of STC buffer (1 M sorbitol, 10 mM Tris pH 7.5, and 10 mM CaCl2).
      5. Resuspend the washed spheroplasts in 50 µL of STC buffer.
        NOTE: Use scissors to cut off the last ~0.7 cm of a pipette tip; this will create a wider mouth and prevent the lysis of the spheroplasts.
    3. Transform the naïve cells with protein.
      1. Transform 50 µL of spheroplast aliquots with 50 µL of donor lysate, 20 µL of salmon sperm DNA (2 mg/mL), and 5 µL of a URA3-marked selection plasmid (e.g., pAG426-GFP)28,33.
      2. Incubate the mix at 25 °C for 30 min.
      3. Centrifuge for 5 min at 600 x g to collect the spheroplasts and re-suspend in 150 µL of SOS-buffer (1 M sorbitol, 7 mM CaCl2, 0.25% yeast extract, and 0.5% peptone).
      4. Recover for 30 min at 30 °C.
      5. Plate the entire culture on SD-URA plates.
      6. Quickly overlay the plated culture with warm (~45 °C) SD-URA containing 0.8% agar.
        Note: This will prevent the spheroplasts from bursting
      7. Incubate the transformation plates for 2-3 days at 30 °C.
      8. Using a toothpick, pick successfully growing cells from the overlaid plate and re-streak to single colonies on SD-URA plates.
      9. Pick dozens of colonies for each and streak on 5-FOA plates to eliminate URA3-marked carrier plasmid.
      10. Pick colonies that grow on the 5-FOA plates and test for the transfer of stable phenotypic states using the growth assay, as before (step 1.13).

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Results

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Protein overexpression is known to dramatically alter cellular phenotypes27. Indeed, with an initial screening approach, hundreds of new phenotypes were reproducibly recovered from the overexpression of clones from the yeast ORFeome using just ten stressors. However, the assays described above allow for the assessment of whether cells retain any long-term stable phenotypes following this overexpression. One protein capable of encoding such a state is Psp1. Psp1 is ...

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Discussion

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The first yeast prions were identified by their unusual phenotypes and perplexing patterns of inheritance. The characteristics of these prions were then used to build algorithms and computational tools to screen for additional prion proteins. The method described here, in contrast, is experimental and relies on transient overexpression to create a lasting change-a stable state-encoded in protein conformation. However, if the efficiency of "seeding" prion assembly by overexpression for any given protein is very lo...

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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We thank Sohini Chakrabortee, Sandra Jones, David Garcia, Bhupinder Bhullar, Amelia Chang, Richard She, and Susan Lindquist for their assistance in developing the assays used in this paper, as well as the reviewers for their thoughtful comments.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Guanidine hydrochlorideSigmaCat#G3272-25GChemical
Manganese chlorideSigmaCat#M8054-100GChemical
Ethidium bromideSigmaE1510Chemical
5-Fluoroorotic AcidSigmaCat#F5013-50MGChemical
BY4741 MATa (his3Δ1 leu2Δ0 LYS2 met15Δ0 ura3Δ0)Winston et al., 1995; Brachmann et al., 1998N/AYeast strain
BY4741 MATα (his3Δ1 leu2Δ0 lys2Δ0 MET15 ura3Δ0)Winston et al., 1995; Brachmann et al., 1998N/AYeast strain
Hsp70 (K69M) Jarosz et al., 2014bN/APlasmid
FLEXGene libraryHu et al., 2007N/APlasmid library
Dextrose (glucose)Fisher ScientificD16-3Media component
RaffinoseSigmaR0250-25GMedia component
GalactoseFisher ScientificBP656-500Media component
CSMSunrise Science1001-100Media component
CSM-URASunrise Science1004-100Media component
CSM-LYSSunrise Science1032-100Media component
CSM-METSunrise Science1019-100Media component
CSM-LYS-METSunrise Science1035-100Media component
yeast extractFisher ScientificBP1422-2Media component
peptoneResearch Products InternationalP20240-5000Media component
bacto-peptoneBD211677Media component
glycerolEMD MilliporeGX0185-2Media component
yeast nitrogen base w/o amino acidsBD291920Media component
agarIBI ScientificIB49172Media component
Adenine sulfateSigmaA3159-25GMedia component
Potassium acetateSigmaP1190-500GMedia component
UracilSigmaU0750-100GMedia component
HistidineSigmaH8000-100GMedia component
LeucineSigmaL8000-25GMedia component
LysineSigmaL5501-25GMedia component
RNase I Thermo Fisher ScientificEN0601Enzyme
biotinylated DNaseThermo Fisher ScientificAM1906Enzyme
zymolyase 100T (yeast lytic enzyme)Sunrise ScienceN0766555Enzyme
Microplate readerBioTekSynergy H1Equipment
Microplate stackerBioTekBioStack3Equipment
Plate fillerBiotTekEL406Equipment
Liquid handling robotBeckman CoulterBiomek FXEquipment

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Protein based InheritanceYeast ORFeome ScreeningHigh throughput PhenotypingPrion Protein DetectionManganese Chloride StressorHSP104 DisaggregasePSP1 Open Reading FrameNon mendelian InheritanceYeast Prion FieldEpigenetic Screening

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